Despite extensive knowledge of the underlying molecular mechanisms that cause cancer, most advanced cancers remain incurable with current chemotherapy and radiation protocols. Oncolytic viruses have emerged as a platform technology that has the potential to significantly augment current standard treatment for a variety of malignancies (Kumar, S. et al., Current opinion in molecular therapeutics 10(4):371-379 (2008); Kim, D. Expert opinion on biological therapy 1(3):525-538 (2001); Kim D. Oncogene 19(56):6660-6669 (2000)). ONYX-015, which has a deletion of the viral E1b-55k gene, was postulated to confer tumor-selective replication in tumors with defects in the p53 pathway (Heise, C. et al., Nat Med 3(6):639-645 (1997); McCormick, F. Oncogene 19(56):6670-6672 (2000); Bischoff, J. R. et al., Science 274(5286):373-376 (1996)). E1b-55k binds and inactivates p53, permitting unscheduled DNA synthesis and viral replication. Inactivation of p53 by E1b-55k, while critical for efficient replication of adenovirus in normal cells, was hypothesized to be irrelevant in tumors having inactivating mutations in the p53 pathway. Preclinical studies demonstrated that a wide range of human tumor cells with mutant or normal p53 gene sequences supported the replication of ONYX-015 in cell culture, demonstrating that permissive viral replication in tumors cells was not strictly dependent on the p53 binding effects of E1b-55k (Heise, C. et al., Nat Med 3(6):639-645 (1997); Heise, C. et al., Clin Cancer Res 6(12):4908-4914 (2000); Rogulski, K. R. et al. Cancer Res 60(5):1193-1196 (2000); Harada, J. N. et al., J Virol 73(7):5333-5344 (1999); Goodrum, F. D. et al., Journal of virology 72(12):9479-9490 (1998)). Previous studies had demonstrated that E1b-55k is a multifunctional protein that, in addition to binding and inactivating p53 during the early phase of infection, facilitates mRNA transport across the nuclear membrane during later phases of infection (Babiss, L. E. et al., Mol Cell Biol 5(10):2552-2558 (1985); Leppard, K. N. et al., Embo J 8(8):2329-2336 (1989)). The lack of efficient mRNA transport due to the deletion of E1b-55k resulted in lower viral replication even in tumor cells when compared to wild-type Ad5. Detailed analysis demonstrated that the loss of the mRNA transport function provided by E1b-55k could be complemented in tumor cell lines, suggesting a novel mechanism of tumor-selective viral replication (O'Shea, C. C. et al., Cancer Cell 8(1):61-74 (2005)). However, complementation of lost functions due to viral gene deletion was incomplete in most tumor cells since the titer of ONYX-015 in tumor cells was often one to two logs lower than wild-type Ad5 in the same tumor cells (Heise, C. et al., Clin Cancer Res 6(12):4908-4914 (2000); Goodrum, F. D. et al., Journal of virology 72(12):9479-9490 (1998)).
Clinical trials using ONYX-015 in a variety of malignancies, including head and neck, colorectal, pancreatic, lung, breast and brain cancer have demonstrated that this oncolytic virus was well tolerated when administered alone or with chemotherapy (Heise, C. et al., Nat Med 3(6):639-645 (1997); Galanis, E. et al., Gene Ther 12(5):437-445 (2005); Chiocca, E. A. et al., Mol Ther 10(5):958-966 (2004); Hecht, J. R. et al., Clin Cancer Res 9(2):555-561 (2003); Reid, T. et al., Cancer Res 62(21):6070-6079 (2002); Vasey, P. A. et al., J Clin Oncol 20(6):1562-1569 (2002); Reid, T. et al., Gene Ther 8(21):1618-1626 (2001); Nemunaitis, J. et al., J Clin Oncol 19(2):289-298 (2001); Kim, D. Gene Ther 8(2):89-98 (2001); Nemunaitis, J. et al., Gene Ther 8(10):746-759 (2001)). However, objective clinical responses following ONYX-015 administration were uncommon raising concerns that this virus, while well tolerated by intratumoral, intravenous and even intraarterial administration, lacked sufficient potency to be broadly applicable as a therapeutic agent (Kim, D. Gene Ther 8(2):89-98 (2001)). The low viral titer for ONYX-015 in various tumor cell lines when compared to wild-type Ad5 raised concerns that the potency of ONYX-015 was not sufficient to be clinically active as an anticancer agent. In addition, E1a, the first protein produced by the virus, was not under tumor-selective control, permitting expression of this potent viral protein in normal cells as well as tumor cells. Since the function of E1a is to facilitate entry of cells into cell division, an oncolytic virus would optimally have both E1a and E1b under tumor-selective control.
Extensive work has been directed at developing a tumor-selective virus with improved anti-tumor potency when compared to ONYX-015. One approach that we and others have taken for making oncolytic viruses has been to insert tumor-selective promoter elements upstream of critical transcription units including E1a, E1b and E4 (Li, Y. et al., Clin Cancer Res 11(24 Pt 1):8845-8855 (2005); Huang, T. G. et al., Gene Ther 10(15):1241-1247 (2003); Wirth, T. et al., Cancer Res 63(12):3181-3188 (2003); Gu, J. et al., Gene Ther 9(1):30-37 (2002); Johnson, L. et al., Cancer Cell 1(4):325-337 (2002); Li, X. et al., Cancer Res 65(5):1941-1951 (2005); Li, Y. et al., Mol Cancer Ther 2(10):1003-1009 (2003)). Incorporation of heterologous promoter elements, such as the promoter for prostate specific antigen (PSA), carcinogenic-embryonic antigen (CEA), E2F1 and telomerase, has achieved variable levels of tumor-selective replication. The potential clinical utility of viruses using heterologous promoters to provide tumor-selective replication has been limited by non-selective and leaky gene expression, diminished capacity of these vectors to replicate when compared to the wild-type virus and recombination events due to the heterologous promoter sequence. For example, ONYX-411 was developed to improve on the potency of ONYX-015 by insertion of the E2F1 promoter region upstream of E1a and E4 (Johnson, L. et al., Cancer Cell 1(4):325-337 (2002)). However, this virus has not been developed for clinical use. In an effort to overcome some of the limitations of heterologous enhancer sequences, we have evaluated the native viral transcriptional control region for E1a to determine if tumor-selective viral replication could be achieved by directed engineering of the native E1a enhancer.